TOR Signaling and Rapamycin Influence Longevity by
Regulating SKN-1/Nrf and DAF-16/FoxO
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Citation
Robida-Stubbs, Stacey, Kira Glover-Cutter, Dudley W. Lamming,
Masaki Mizunuma, Sri Devi Narasimhan, Elke NeumannHaefelin, David M. Sabatini, and T. Keith Blackwell. “TOR
Signaling and Rapamycin Influence Longevity by Regulating
SKN-1/Nrf and DAF-16/FoxO.” Cell Metabolism 15, no. 5 (May
2012): 713–724. © 2012 Elsevier Inc.
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http://dx.doi.org/10.1016/j.cmet.2012.04.007
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Cell Metabolism
Article
TOR Signaling and Rapamycin Influence Longevity
by Regulating SKN-1/Nrf and DAF-16/FoxO
Stacey Robida-Stubbs,1 Kira Glover-Cutter,1 Dudley W. Lamming,2,3,4 Masaki Mizunuma,1,5 Sri Devi Narasimhan,1
Elke Neumann-Haefelin,1,6 David M. Sabatini,2,3,4 and T. Keith Blackwell1,*
1Joslin
Diabetes Center, Harvard Stem Cell Institute, and Department of Genetics, Harvard Medical School, Boston, MA 02215, USA
Institute for Biomedical Research, Cambridge MA 02142, USA
3Department of Biology, Howard Hughes Medical Institute, and David H. Koch Institute for Integrative Cancer Research,
Massachusetts Institute of Technology, Cambridge, MA 02139, USA
4Broad Institute of Harvard and MIT, 7 Cambridge Center, Cambridge, MA 02142, USA
5Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University,
Higashi-Hiroshima 739-8530, Japan
6Renal Division, University Hospital Freiburg, 79106 Freiburg, Germany
*Correspondence: keith.blackwell@joslin.harvard.edu
DOI 10.1016/j.cmet.2012.04.007
2Whitehead
SUMMARY
The TOR kinase, which is present in the functionally
distinct complexes TORC1 and TORC2, is essential
for growth but associated with disease and aging.
Elucidation of how TOR influences life span will identify mechanisms of fundamental importance in aging
and TOR functions. Here we show that when TORC1
is inhibited genetically in C. elegans, SKN-1/Nrf, and
DAF-16/FoxO activate protective genes, and increase stress resistance and longevity. SKN-1 also
upregulates TORC1 pathway gene expression in a
feedback loop. Rapamycin triggers a similar protective response in C. elegans and mice, but increases
worm life span dependent upon SKN-1 and not
DAF-16, apparently by interfering with TORC2
along with TORC1. TORC1, TORC2, and insulin/
IGF-1-like signaling regulate SKN-1 activity through
different mechanisms. We conclude that modulation
of SKN-1/Nrf and DAF-16/FoxO may be generally
important in the effects of TOR signaling in vivo
and that these transcription factors mediate an
opposing relationship between growth signals and
longevity.
INTRODUCTION
The TOR (target of rapamycin) kinase integrates nutrient and
anabolic signals to promote growth (Ma and Blenis, 2009; Wullschleger et al., 2006; Zoncu et al., 2011). TOR is found in two
distinct complexes, TORC1 and TORC2 (Figure S1 available
online). Amino acid, oxygen, energy, and growth signals activate
TORC1, which phosphorylates a set of well-characterized substrates to increase messenger RNA (mRNA) translation and
inhibit autophagy. The amino acid signal is transduced by heterodimeric Rag GTPases that recruit TORC1 to lysosomal membranes, where it is activated by Rheb. Growth signals and
interaction with the ribosome activate TORC2, which in turn activates AKT, SGK, and related kinases (Oh et al., 2010; Zinzalla
et al., 2011; Zoncu et al., 2011).
TOR signaling is essential for growth and development,
but has also been implicated in diabetes, cardiac hypertrophy,
malignancies, neurodegenerative syndromes, other diseases,
and aging (Stanfel et al., 2009; Wullschleger et al., 2006; Zoncu
et al., 2011). TOR inhibitors have been approved or are under
investigation for treatment of several conditions, including
various cancers. These inhibitors include the immunosuppressant rapamycin, which is widely used to combat kidney
rejection after transplantation. In model organisms ranging
from yeast to mice, inhibition of TOR signaling increases life
span (Bjedov et al., 2010; Harrison et al., 2009; Kapahi et al.,
2010; Kenyon, 2010; Selman et al., 2009; Stanfel et al., 2009).
TOR has also been implicated in dietary restriction (DR), an intervention that extends life span and protects against chronic
disease. Rapamycin increases mouse life span even when
administered late in life (Harrison et al., 2009), suggesting that
pharmacological targeting of TOR signaling might be a promising
antiaging strategy. However, rapamycin is associated with
insulin resistance as well as immunosuppression (Zoncu et al.,
2011; Lamming et al., 2012), making it important to identify
specific mechanisms downstream of rapamycin that affect
aging.
TOR inhibition, rapamycin, and DR seem to promote longevity
at least in part by reducing mRNA translation (Bjedov et al., 2010;
Kapahi et al., 2010; Kenyon, 2010; Stanfel et al., 2009; Zid et al.,
2009). A lower level of translation might be beneficial simply
because the burden of protein synthesis is decreased, but recent
evidence indicates that when translation is reduced protective
mechanisms are mobilized, through translation of particular
genes being preserved or even increased (Kapahi et al., 2010;
Rogers et al., 2011; Stanfel et al., 2009; Zid et al., 2009). Genetic
interference with translation initiation increases life span in
C. elegans, and some studies indicate that this effect requires
the conserved transcription factors DAF-16/FoxO (Hansen
et al., 2007; Henderson et al., 2006; Rogers et al., 2011;
Tohyama et al., 2008; Wang et al., 2010) and SKN-1/Nrf (Wang
et al., 2010). This suggests that the benefits of reduced protein
Cell Metabolism 15, 713–724, May 2, 2012 ª2012 Elsevier Inc. 713
Cell Metabolism
SKN-1, DAF-16, and TOR-Associated Longevity
Figure 1. Genetic TORC1 Inhibition Increases Stress Resistance through SKN-1 and DAF-16
(A) Increased autophagy after TORC1-pathway gene knockdown. LGG-1::GFP puncta were counted in seam cells (n) in day 3 adults. ***p % 0.0001, **p < 0.001,
unpaired t test.
(B) Decreased protein synthesis after genetic TORC1 inhibition or rapamycin treatment. *p % 0.005, Student’s one-sided t test. Error bars represent ± SEM.
(C) TORC1 inhibition by ragc-1 RNAi increased oxidative stress (TBHP) resistance dependent upon skn-1 but not daf-16. The skn-1(zu67) and daf-16(mgDf47)
alleles were analyzed in all experiments unless otherwise indicated. In all survival plots, ext. refers to mean survival extension associated with the indicated
intervention, and WT to the wild-type. The y axis indicates proportion surviving.
(D) Increased resistance to heat (35 C) is mediated by both skn-1 and daf-16.
Representative experiments are shown in (A)–(D). See also Tables S1 and S2 for statistics and stress resistance analyses of additional TORC1 pathway genes and
Figure S1.
synthesis might also involve modulation of transcription. DAF-16
and SKN-1 regulate genes that protect against environmental,
metabolic, and proteotoxic stress, and promote longevity in
various species (Kenyon, 2010; Kwon et al., 2010; Li et al.,
2011; Oliveira et al., 2009; Sykiotis and Bohmann, 2010; Tullet
et al., 2008). In C. elegans, they are each inhibited by insulin/
IGF-1-like signaling (IIS), another growth-related pathway that
influences aging across metazoa (Kenyon, 2010; Tullet et al.,
2008).
The genetic evidence that DAF-16 and SKN-1 are important
for the benefits of reducing translation raises the question of
whether TOR signaling might actually regulate these transcription factors. It is unknown whether TOR affects SKN-1/Nrf
activity, but it is generally accepted that TOR influences aging
by acting independently from DAF-16/FoxO and IIS, in large
part because daf-16 is not needed for life span to be extended
by reduced TOR kinase (LET-363) activity or most DR regimens
(Bishop and Guarente, 2007; Hansen et al., 2007; Honjoh et al.,
2009; Kapahi et al., 2010; Kenyon, 2010; Panowski et al., 2007;
Sheaffer et al., 2008; Vellai et al., 2003). Lack of the TOR kinase
would eliminate both TORC1 and TORC2, however, making it
critical to establish how TORC1 and TORC2 affect longevity
independently of each other.
Here we have investigated how longevity is affected by genetic
TORC1 or TORC2 inhibition and, for the first time in C. elegans,
rapamycin. We find that life span is increased in a SKN-1-depen714 Cell Metabolism 15, 713–724, May 2, 2012 ª2012 Elsevier Inc.
dent manner when either TOR pathway is inhibited. DAF-16 is
also required for life span to be extended by genetic inhibition
of TORC1, but not TORC2. Genetic interference with TORC1
results in a SKN-1- and DAF-16-mediated transcriptional response that may be triggered by lower levels of translation.
Rapamycin induces a similar response but extends life span
independently of daf-16, apparently by inhibiting both TORC1
and TORC2. We conclude that both SKN-1/Nrf and DAF-16/
FoxO are opposed by TOR signaling, and have a central role in
its influence on aging.
RESULTS
SKN-1/Nrf and DAF-16/FoxO Are Required for Stress
Resistance and Longevity from Reduced TORC1 Activity
To inhibit TORC1 but not TORC2, and to obtain results that were
not confounded by its developmental functions (Jia et al., 2004;
Schreiber et al., 2010), we used RNA interference (RNAi)
to knock down the TORC1-specific genes daf-15 (Raptor) and
rheb-1 (Rheb), and the conserved Rag GTPases raga-1 and
ragc-1 (Figure S1) only during adulthood. We monitored
TORC1 activity by examining autophagy and translation. The
GFP-fused vacuolar protein LGG-1 marks autophagic vesicles.
LGG-1 puncta were increased by knockdown of the insulin
receptor DAF-2, as described (Meléndez et al., 2003), and by
ragc-1 or daf-15 RNAi, as predicted (Figures 1A and S1). In
Cell Metabolism
SKN-1, DAF-16, and TOR-Associated Longevity
contrast, autophagy was reduced by knockdown of the S6
kinase RSKS-1, which increases translation downstream of
TORC1 but also promotes autophagy (Scott et al., 2004). As expected, ragc-1 RNAi decreased overall mRNA translation, as
measured by 35S methionine incorporation (Figure 1B).
Knockdown of each TORC1 pathway gene that we examined
(raga-1, ragc-1, rheb-1, daf-15) dramatically enhanced stress
tolerance. skn-1 but not daf-16 was required for increased resistance to the oxidizing agent tert-butyl hydrogen peroxide (TBHP)
(Figure 1C and Table S1). This result mimicked the effect of inhibiting translation initiation (Wang et al., 2010). Interference with
TORC1 by ragc-1 RNAi increased heat resistance in daf-16
and skn-1 mutants, but not a skn-1; daf-16 double mutant, indicating involvement of both daf-16 and skn-1 (Figure 1D and
Table S2). The increases in stress resistance that result from
genetic TORC1 inhibition are therefore mediated by both
SKN-1 and DAF-16, with SKN-1 being essential under TBHP
oxidative stress conditions.
Adulthood knockdown of each TORC1 pathway gene that we
tested increased life span (Figures 2A and 2B, Tables S3 and S4).
Reduced TORC1 activity also delayed the age-associated
decline in two health span indicators, fast movements and
pharyngeal pumping, indicating that aging was slowed (Figures
2C and 2D). In most experiments, knockdown of TORC1
pathway genes failed to increase life span in a skn-1 mutant (Figure 2A and Tables S3 and S4). Surprisingly, RNAi against these
four genes also did not increase life span in daf-16 mutants (Figure 2B and Tables S3 and S4), in contrast to the daf-16-independent longevity associated with TOR kinase inhibition (Hansen
et al., 2007; Sheaffer et al., 2008; Vellai et al., 2003). We obtained
similar findings with or without inclusion of fluorodeoxyuridine
(FUdR) to prevent offspring production (Tables S3 and S4). Life
span of the skn-1; daf-16 mutant was unaffected by raga-1
RNAi but increased by inhibition of mitochondrial genes (cco-1,
cyc-1; Tables S3 and S4), which involves a distinct longevity
pathway (Durieux et al., 2011; Kenyon, 2010). This indicates
that animals that lack SKN-1 and DAF-16 are not simply refractory to life span extension. Both SKN-1 and DAF-16 are therefore
required for the delays in aging that result from genetic TORC1
inhibition.
Somatic Effects of TORC1 on Longevity
Given that TORC1 promotes protein synthesis, and that inhibition of translation reduces fecundity (Hansen et al., 2007; Pan
et al., 2007), it is possible that TORC1 inhibition might increase
longevity indirectly, through effects on the germline. Reductions
in germline stem cell (GSC) number delay aging, presumably to
preserve the organism during adversity (Kenyon, 2010). When
the number of C. elegans GSCs is reduced, DAF-16 accumulates
in intestinal nuclei. The accompanying longevity extensions
require DAF-16 and the ankyrin repeat protein KRI-1 (Kenyon,
2010).
Several lines of evidence indicate that genetic interference
with TORC1 or translation extends life span independently of
the GSC pathway. Fecundity and presumably GSC proliferation
were not impaired by adulthood knockdown of TORC1 pathway
genes (Figure 2E). Knockdown of raga-1 or the translation initiation factors ifg-1 and eif-1 increased life span in the temperaturesensitive (ts) germline-defective mutant glp-1(bn18), under
conditions where germ cell proliferation was blocked and life
span was extended (Figure 2F and Tables S3 and S4). Finally,
knockdown of either raga-1 or eif-1 extended the life span of
kri-1 mutants (Figure 2G and Tables S3 and S4), in which the
GSC pathway is blocked (Kenyon, 2010). In C. elegans, tissuespecific RNAi can be performed in strains where the RNAi-defective mutation rde-1 is rescued in individual tissues (Qadota et al.,
2007). ragc-1 RNAi increased life span in a strain in which RNAi is
restricted to the intestine (VP288; Figure 2H and Tables S3
and S4). This suggests that aging is delayed when amino acid
signaling to TORC1 is impaired specifically in the intestine, which
corresponds to the mammalian liver, gut, and adipose tissues
and plays a central role in metabolism and longevity (Kenyon,
2010).
TORC1 Inhibits SKN-1/Nrf-and DAF-16/FoxO-Driven
Transcription
Our results raise an important question: are activities of SKN-1
and DAF-16 actually controlled by TORC1, or does longevity
associated with reduced TORC1 activity simply depend upon
constitutive functions of these transcription factors? The finding
that TORC1 regulates SKN-1 and DAF-16 would strongly
support the model that they play central roles in its effects on
aging. When translation initiation factors are knocked down by
RNAi, reporters in which promoters of the conserved SKN-1/
Nrf targets gcs-1 and gst-4 are fused to GFP (gcs-1p::GFP and
gst-4::GFP) are activated in the intestine, and several SKN-1
target genes are upregulated (Wang et al., 2010). If SKN-1
targets are activated by interference with translation, then
genetic inhibition of TORC1 might induce a similar response.
Accordingly, knockdown of TORC1 pathway genes increased
intestinal gcs-1p::GFP and gst-4::GFP expression (Figures 3A,
S2A, and S2B). We next analyzed how expression of endogenous SKN-1 target genes (Oliveira et al., 2009; Wang et al.,
2010) was affected when TORC1 was inhibited through Rag
gene RNAi (Figures 3B, S2C, and S2D). These and other
SKN-1 targets that we analyzed in this study represent various
stress-defense functions (glutathione synthesis, glutathione
S-transferase, ABC transporter, oxidoreductase, lysosomal protease). Rag gene RNAi increased expression of these genes in
a skn-1-dependent manner, but did not activate control genes
(Figures 3B, S2C, and S2D). We conclude that when TORC1 is
inhibited, SKN-1 induces a protective transcriptional response.
To investigate further whether TORC1 regulates transcription
of SKN-1 target genes, we used chromatin immunoprecipitation
(ChIP) to detect endogenous SKN-1 and markers of transcription
activity at the SKN-1 targets gst-10, sdz-8, and gcs-1. TORC1
inhibition (raga-1 RNAi) increased expression of these genes
(Figures 3C and S2E), accompanied by binding of endogenous
SKN-1 to their regulatory regions (Figures 3D and S2F). During
transcription initiation, RNA Polymerase II (Pol II) is phosphorylated on Ser 5 of its C-terminal domain (CTD) repeat (P-Ser5)
(Bentley, 2005). Phosphorylation of the CTD repeat on Ser 2
(P-Ser2) also marks transcription, particularly the elongation
step. TORC1 inhibition significantly increased P-Ser5 levels 50
of the gcs-1 coding region (Figure S2G). At gst-10 and sdz-8
we assayed P-Ser2, levels of which were similarly increased (Figure 3E). Together, the data indicate that TORC1 suppresses
SKN-1-mediated transcription of protective genes.
Cell Metabolism 15, 713–724, May 2, 2012 ª2012 Elsevier Inc. 715
Cell Metabolism
SKN-1, DAF-16, and TOR-Associated Longevity
Figure 2. Life Span Extensions from Genetic TORC1 Inhibition Require SKN-1 and DAF-16
(A and B) skn-1 (A) and daf-16 (B) are required for genetic TORC1 inhibition to increase longevity.
(C and D) Rag GTPase knockdown increases health span. raga-1 or ragc-1 RNAi preserves fast body movements (C) and fast pharyngeal pumping (D). **p %
0.008, log rank for (C); **p < 0.007, *p < 0.08, log rank for (D).
(E) Brood size is unaffected by adulthood TORC1 pathway gene RNAi or rapamycin. n = 3–7 worms. Error bars represent ± SEM.
(F) Genetic TORC1 inhibition extends life span in glp-1(ts) animals independently of the GCS pathway. WT or glp-1(ts) animals were placed at the nonpermissive
temperature (25 C) from the L2 stage until adulthood, then maintained at 20 C, a protocol that prevents germ cell proliferation in glp-1(ts). raga-1 or control RNAi
was initiated at the beginning of adulthood.
(G) TORC1 inhibition by raga-1 RNAi extends life span in kri-1(ok1251) mutants, in which germ cell arrest fails to extend life span.
(H) TORC1 inhibition by intestinal ragc-1 RNAi. In VP288, rde-1 is rescued using the intestine-specific promoter nhx-2 (Durieux et al., 2011; Qadota et al., 2007).
Survival plots show composite or individual experiments that were performed in parallel. See also Table S3 for corresponding data, analyses of additional TORC1
pathway genes, and statistics, and Table S4 for individual experiments.
A genome-scale ChIP analysis revealed that transgenically expressed SKN-1 is also present at potential regulatory regions of
the TORC1 pathway genes daf-15, rsks-1, raga-1, and ragc-1
(Niu et al., 2010), suggesting that SKN-1 and TORC1 might regulate their expression. Supporting this idea, ragc-1 RNAi led to an
increase in TORC1 pathway gene expression that was largely
skn-1-dependent (Figure 3F). A reduction in TORC1 signaling
716 Cell Metabolism 15, 713–724, May 2, 2012 ª2012 Elsevier Inc.
therefore directs SKN-1 not only to activate protective genes,
but also to increase TORC1 pathway gene expression in a feedback loop.
We also investigated whether DAF-16 might be regulated by
TORC1 and translation inhibition. Knockdown of either Rag gene
led to daf-16-dependent activation of the conserved DAF-16/
FoxO target sod-3 (superoxide dismutase) in the intestine and
Cell Metabolism
SKN-1, DAF-16, and TOR-Associated Longevity
Figure 3. Genetic TORC1 Inhibition Induces SKN-1- and DAF-16-Mediated Transcription
(A) skn-1-dependent induction of the SKN-1 target promoter gcs-1 by Rag GTPase RNAi. ***p < 0.0001, **p < 0.001, chi2 method. L, low; M, medium; H, high.
Scoring is described in the Experimental Procedures.
(B) Activation of endogenous SKN-1 target genes by ragc-1 RNAi, measured by quantitative RT-PCR (qRT-PCR) in WT animals, or skn-1 mutants. Fold induction
relative to WT vector control is shown for all qRT-PCR data.
(C–E) Transcriptional activation of the SKN-1 targets gst-10 and sdz-8 by raga-1 RNAi, detected by qRT-PCR (C) in lysates that were analyzed by ChIP in
(D and E). The relative ChIP signal is shown for endogenous SKN-1 (D) and Serine 2-phosphorylated Pol II CTD (Ser2) (E) along each gene. Positions indicated
below the graphs correspond to the middle of each qPCR amplicon relative to the predicted transcription start site. The percent ChIP signal is relative to input and
normalized to the highest signal in each qPCR run (Glover-Cutter et al., 2008). Analyses of intergenic regions and control genes (data not shown) indicated that
signals of 25% and 10% represent thresholds for specific presence of SKN-1 and P-Ser2, respectively.
(F) Activation of endogenous TORC1 pathway genes by ragc-1 RNAi.
(G) Induction of endogenous DAF-16 target genes in response to ragc-1 RNAi. ***p < 0.001, **p < 0.01, *p < 0.05; NS, not significant. All qRT-PCR and ChIP
p values in this study were calculated by one- or two-sided t test, as appropriate.
Error bars represent ± SEM. See also Figure S2.
to upregulation of multiple endogenous DAF-16 target genes
(Kwon et al., 2010; Murphy et al., 2003) (Figures 3G, S2H, and
S2I). These DAF-16 targets included small heat-shock protein
genes that have been implicated in longevity (sip-1, hsp-16.1,
hsp-12.6). Knockdown of the translation factor ifg-1 also activated many of these endogenous DAF-16 target genes, consistent with the idea that translation suppression is involved (Figure S2J). DAF-16 reduced expression of rsks-1 and daf-15,
consistent with a previous report (Jia et al., 2004), and was not
required for genetic TORC1 inhibition to increase raga-1 expression (Figure 3F), indicating a distinct function from SKN-1 in
TORC1 pathway autoregulation. The data indicate that both
SKN-1 and DAF-16 induce protective gene expression when
TORC1 activity is reduced, and therefore that TORC1 signaling
opposes each of these two transcription factors.
Distinct Transcriptional Responses Regulated
by TORC1 and IIS
The essential role played by SKN-1 in TORC1-associated life
span extension was surprising, because SKN-1 is not as impor-
tant as DAF-16 for longevity that is associated with reduced IIS
(Tullet et al., 2008). Our results could be explained if TORC1
and IIS have distinct effects on SKN-1, DAF-16, and their
downstream target genes. IIS inhibits SKN-1 and DAF-16
through phosphorylation, so that they accumulate in nuclei
when IIS is reduced (Kenyon, 2010; Tullet et al., 2008) (i.e.,
daf-2 RNAi, Figures 4A–4C). In contrast, SKN-1 generally
did not accumulate in intestinal nuclei in response to genetic
inhibition of either TORC1 (Figures 4A, S3A, and S3B), or translation initiation (Wang et al., 2010). TORC1 might influence
whether SKN-1 that is already in the nucleus is recruited to
promoters, a mechanism that is feasible because SKN-1
occupies many promoters even under nonstressed conditions
(Niu et al., 2010). Multiple DAF-16 isoforms accumulate in
nuclei when IIS is reduced (Kenyon, 2010; Kwon et al., 2010),
but only a single DAF-16 isoform (DAF-16f) localized to intestinal nuclei after genetic inhibition of TORC1 or translation initiation (Figures 4B, 4C, and S3C–S3F). This DAF-16 isoform
appears to be particularly important for longevity (Kwon et al.,
2010).
Cell Metabolism 15, 713–724, May 2, 2012 ª2012 Elsevier Inc. 717
Cell Metabolism
SKN-1, DAF-16, and TOR-Associated Longevity
Figure 4. Distinct Transcriptional Responses to Inhibition of TORC1 and IIS
(A) Genetic TORC1 inhibition generally does not increase intestinal SKN-1 nuclear occupancy. SKN-1B/C::GFP encodes two of three SKN-1 isoforms (An and
Blackwell, 2003). Scoring is described in the Experimental Procedures.
(B) Analysis of DAF-16 nuclear occupancy in the intestine. This transgene encodes the DAF-16a isoform (Henderson et al., 2006).
(C) Accumulation of DAF-16f (Kwon et al., 2010) in intestinal nuclei after genetic inhibition of TORC1 or translation.
(D) Transcriptional activation of certain genes after genetic inhibition of TORC1 but not IIS, analyzed by qRT-PCR.
***p < 0.0001; NS, not significant; chi2 method; L, low; M, medium; H, high for (A)–(C). ***p < 0.001, **p < 0.01, *p < 0.05 for (D). Error bars represent ± SEM. See
also Figure S3.
Genetic inhibition of TORC1 or translation initiation also
induced transcription of various genes that are not activated
by reduced IIS. These included skn-1 and daf-16 themselves,
the superoxide dismutase sod-4, which is upregulated by DR
(Panowski et al., 2007), and the insulin peptide ins-7, which is
inhibited by SKN-1 and DAF-16 under normal and reduced IIS
conditions, respectively (Murphy et al., 2003; Oliveira et al.,
2009) (Figures 4D and S3G). We conclude that TORC1 and IIS
act through different mechanisms to regulate SKN-1 and
DAF-16, and thereby modulate transcription of overlapping but
distinct sets of downstream genes.
SKN-1/Nrf- and DAF-16/FoxO-Mediated Transcriptional
Responses to Rapamycin
Rapamycin has not been reported to affect C. elegans longevity
or stress defense, but our findings provided a readout of TORC1
inhibition that allowed us to optimize rapamycin concentration.
Exposure of adults to 100 mM rapamycin upregulated gcs1p::GFP in a skn-1-dependent manner, and reduced translation
(Figures 1B, 5A, and S4A). This rapamycin concentration is
higher than that used in cultured cells, but high concentrations of
compounds are often required for bioavailability in C. elegans
(Kokel et al., 2006). Importantly, rapamycin treatment upregu718 Cell Metabolism 15, 713–724, May 2, 2012 ª2012 Elsevier Inc.
lated genes that were activated by genetic TORC1 inhibition,
including skn-1, daf-16, and genes that encode TORC1 pathway
components (Figures 5B–5E, S4B, and S4C). Rapamycin did
not interfere with bacterial proliferation, indicating that these
effects did not derive artifactually from food limitation (Figure S4D). As seen with genetic TORC1 inhibition, rapamycin
did not increase the levels of SKN-1 in nuclei and affected only
the DAF-16f isoform (Figures 5F, S4E, and S4F). SKN-1 target
activation was accompanied by increased presence of SKN-1
and transcription markers at these genes, however, indicating
direct regulation by SKN-1 (Figures 5G–5I and S4G–S4L). Rapamycin-induced activation of SKN-1 target genes was, in general,
SKN-1 dependent (Figure 5B), and DAF-16 target gene induction
was largely abolished in a daf-16 mutant (Figure 5E). We
conclude that rapamycin induces transcription of SKN-1- and
DAF-16-regulated protective genes.
We investigated whether the transcriptional responses to
rapamycin we have detected might be conserved in mammals.
Rapamycin-treated or control mice were fasted overnight to
control for feeding status and its effects on TORC1, and then
were either refed or not. In the liver, rapamycin increased expression of the FoxO target G6Pase under refeeding but not
fasting conditions, as described (Figures 5J and 5K) (Lamming
Cell Metabolism
SKN-1, DAF-16, and TOR-Associated Longevity
Figure 5. Rapamycin Activates SKN-1- and DAF-16-Mediated Transcription
(A) Activation of gcs-1::GFP by rapamycin. 100 mM (in the agar) was the lowest concentration at which near-maximal induction occurred. ***p < 0.0001; NS, not
significant; chi2 method. L, low; M, medium; H, high.
(B–E) Rapamycin-induced activation of endogenous SKN-1 target (B), TORC1 pathway (C), other TORC1-regulated (D), and DAF-16 target (E) genes, analyzed by
qRT-PCR.
(F) Rapamycin does not induce nuclear accumulation of SKN-1B/C::GFP in worms grown on OP50.
(G–I) Activation of SKN-1 target gene transcription by rapamycin, with accumulation of SKN-1 (H) and Serine 2-phosphorylated Pol II CTD (Ser2) (I) detected by
ChIP as in Figures 3C–3E.
(J and K) Rapamycin increases Nrf/SKN-1 target gene expression in mouse liver. Vehicle- or rapamycin-treated male mice were fasted (K), or fasted then refed (J)
(n = 5 for each) prior to analysis of RNA by qRT-PCR. Genes that are regulated by Nrf1 (Mt1) (Ohtsuji et al., 2008) or Nrf2 (others) (Malhotra et al., 2010; Wu et al.,
2011) were assayed along with the FoxO target G6Pase (Mihaylova et al., 2011).
***p < 0.001, **p < 0.01, *p < 0.05; NS, not significant (B–K). Error bars represent ± SEM. See also Figure S4.
et al., 2012). Similarly, in the refed group, rapamycin upregulated
most of the ten Nrf/SKN-1 targets we tested, each of which is
involved in stress defense, with five genes reaching statistical
significance (Figure 5J). These effects were also largely attenuated by fasting (Figure 5K). We conclude that rapamycin treatment activates SKN-1/Nrf-regulated protective genes in vivo in
mammals, as we observed in C. elegans.
SKN-1/Nrf but Not DAF-16/FoxO Is Required for
Longevity from Rapamycin or TORC2 Inhibition
Exposure of adult C. elegans to rapamycin dramatically increased both stress resistance and life span. Rapamycin increased oxidative stress (TBHP) resistance dependent upon
skn-1 but not daf-16, and increased life span in a skn-1-depen-
dent manner, as seen with genetic TORC1 inhibition (Figure 6A
and Tables S1 and S5). In contrast, rapamycin robustly increased life span in two daf-16 mutants (mgDf47 and mu86).
Similar results were obtained with or without FUdR, and with
growth on either the standard strain OP50 or the feeding RNAi
strain HT115 (Table S5). The daf-16 independence of longevity
deriving from adulthood rapamycin treatment, together with
our finding that rapamycin did not impair fecundity (Figure 2E),
argued that rapamycin does not increase life span through the
GSC pathway.
Our finding that rapamycin increased life span independently
of daf-16 indicates that rapamycin influences an additional
longevity pathway besides TORC1, and parallels previous analyses of the TOR kinase, in which daf-16 was not required
Cell Metabolism 15, 713–724, May 2, 2012 ª2012 Elsevier Inc. 719
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SKN-1, DAF-16, and TOR-Associated Longevity
Figure 6. Rapamycin-Induced Life Span Extension May Involve TORC1 and TORC2
(A) Longevity from rapamycin requires skn-1 but not daf-16.
(B and C) TORC2 inhibition (rict-1 RNAi) increases life span dependent upon skn-1 (B) but not daf-16 (C).
(D) rict-1 RNAi extends life span in VP288, in which RNAi is active specifically in the intestine (see the main text). Representative (rapamycin) or composite (rict-1)
experiments are shown, with statistics and additional analyses presented in Tables S6 and S7.
(E) TORC2 or TOR kinase (let-363) inhibition by feeding RNAi increases SKN-1 nuclear occupancy.
(F and G) SKN-1 nuclear accumulation in rict-1 mutants (F) and after rapamycin treatment (G) depends upon the food source.
***p < 0.0001; NS, not significant; chi2 method. L, low; M, medium; H, high. Error bars represent ± SEM. See also Tables S5–S7.
(Hansen et al., 2007; Sheaffer et al., 2008; Vellai et al., 2003).
Although rapamycin is generally considered to be a TORC1
inhibitor, in some mammalian cell lines prolonged rapamycin
treatment also interferes with TORC2, apparently by physically
disrupting the TORC2 complex (Zoncu et al., 2011). Recently
rapamycin has also been observed to disrupt TORC2 in vivo in
multiple tissues in the mouse (Lamming et al., 2012). Our results
could be reconciled with the C. elegans TOR kinase literature if
(1) rapamycin interferes with both TORC1 and TORC2 in
C. elegans, as would be the case for TOR loss, (2) TORC2 inhibition increases life span, and (3) the life span extension associated with TORC2 inhibition requires skn-1 but not daf-16.
720 Cell Metabolism 15, 713–724, May 2, 2012 ª2012 Elsevier Inc.
C. elegans with mutations in the TORC2 complex gene rict-1
(Rictor, Figure S1) grow slowly and have a small body size, and
live slightly longer than WT when maintained on ‘‘rich’’ food
such as the RNAi feeding strain HT115 and at elevated temperature (25 C) (Soukas et al., 2009). We investigated whether
TORC2 inhibition might increase longevity at a lower temperature (20 C), and when TORC2 activity was reduced by adulthood
rict-1 RNAi, a strategy that would bypass developmental functions of TORC2. Under these conditions, rict-1 RNAi increased
life span substantially, dependent upon skn-1 (Figure 6B and
Tables S6 and S7). Importantly, daf-16 was not required for life
span to be increased by rict-1 RNAi, or when we blocked both
Cell Metabolism
SKN-1, DAF-16, and TOR-Associated Longevity
TORC1 and TORC2 by ragc-1; rict-1 RNAi (Figure 6C and Tables
S6 and S7). Our results suggest that the daf-16-independent
longevity associated with TOR kinase (let-363) loss (Hansen
et al., 2007; Sheaffer et al., 2008; Vellai et al., 2003) is mediated
by lack of TORC2, not TORC1 and that the effects of rapamycin
we observed may also involve inhibition of both TORC1 and
TORC2. rict-1 RNAi extended life span in the intestine-specific
RNAi strain VP288 (Figure 6D and Tables S6 and S7), indicating
that TORC2, like TORC1, modulates life span at least in part by
acting in the intestine.
Given the importance of SKN-1 for the life span increases
associated with TORC2 inhibition, we investigated whether
TORC2 influences SKN-1 nuclear accumulation. Genetic evidence suggests that in C. elegans, as in mammals, TORC2
increases activity of the AKT and SGK-1 kinases, which phosphorylate SKN-1 and inhibit its accumulation in intestinal nuclei
(Figure S1) (Soukas et al., 2009; Tullet et al., 2008). Accordingly,
SKN-1 accumulated in intestinal nuclei after rict-1 RNAi (Figure 6E). This result contrasted sharply with the failure of rapamycin to increase SKN-1 nuclear accumulation (Figure 5F), but in
those earlier rapamycin experiments worms had been grown
on OP50, on which the life span of rict-1 mutants is decreased
(Soukas et al., 2009). Strikingly, either rict-1 mutation or rapamycin dramatically increased SKN-1 nuclear accumulation when
worms were grown on rich food (HT115 or HB101), but not
OP50 (Figures 6F and 6G). This differs from the effect of daf-2
mutation (reduced IIS), which increases SKN-1 nuclear accumulation in worms maintained on OP50 (Tullet et al., 2008). In
addition, the longevity and life span extensions associated with
rapamycin seemed to be more robust on HT115 than OP50
(Table S5). Finally, RNAi knockdown of the TOR kinase let-363
also increased SKN-1 nuclear accumulation, as would be predicted for an effect on TORC2 (Figure 6E). Taken together, our
experiments suggest that rapamycin promotes longevity by
interfering with both TORC1 and TORC2, with the resulting
effects on SKN-1/Nrf being essential.
DISCUSSION
Our C. elegans experiments have uncovered an important mechanism of TORC1 action: TORC1 opposes both SKN-1/Nrf and
DAF-16/FoxO, so that when TORC1 is inhibited, these factors
increase transcription of protective genes. Previous work had
identified effects of TORC1 inhibition that may enhance longevity, including decreased protein synthesis, preservation of
protective gene translation, and increased autophagy (Bjedov
et al., 2010; Kapahi et al., 2010; Kenyon, 2010; Stanfel et al.,
2009; Zid et al., 2009). Here we have shown that these
TORC1-associated mechanisms fail to increase C. elegans life
span in the absence of either SKN-1 or DAF-16, and therefore
that the SKN-1- and DAF-16-dependent transcription responses
we detected are essential effectors of the longevity that results
from genetic TORC1 inhibition.
We were surprised to find that DAF-16 is required for genetic
TORC1 inhibition to extend life span (Figure 2B and Tables S3
and S4), given that lack of the TOR kinase extends life span independently of DAF-16 (see the Introduction). It has been reported
that daf-16 is needed for life span extensions associated with
daf-15 heterozygosity or raga-1 mutation (Jia et al., 2004;
Schreiber et al., 2010), but several considerations made it
unclear how to interpret those findings: these mutations impair
larval development, DAF-16 inhibits daf-15 expression, and
longevity from TOR kinase loss does not require daf-16. Our
observation that TORC2 inhibition increases life span independently of daf-16 (Figure 6C and Tables S6 and S7) reconciles
these seemingly conflicting studies with each other and our
extensive data on TORC1. It also raises the intriguing possibility
that the daf-16-independent longevity associated with many
C. elegans DR regimens (see the Introduction) might involve
TORC2, especially considering that many rict-1 phenotypes
are influenced by the food source (Figure 6F) (Soukas et al.,
2009). C. elegans DR has been observed to require neuronal
SKN-1 expression and the FoxA transcription factor PHA-4,
which is also needed for life span to be extended by TOR kinase
inhibition (Bishop and Guarente, 2007; Panowski et al., 2007;
Sheaffer et al., 2008). Perhaps some differences among DR protocols could be explained if SKN-1, DAF-16, and possibly PHA-4
have some overlapping functions and are affected differently by
these DR regimens.
It was particularly striking that SKN-1/Nrf was required for life
span extensions associated with genetic inhibition of either
TORC1 or TORC2, or rapamycin treatment, and therefore must
be central to the influence of TOR pathways on aging (Figures
2A, 6A, 6B, and 7 and Tables S3–S7). In contrast, SKN-1 is
only partially required for life span extension associated with
reduced IIS, in which daf-16 is essential (Kenyon, 2010; Tullet
et al., 2008). We have determined that TORC1 and IIS not only
act through different mechanisms to regulate SKN-1 and DAF16, but also regulate expression of overlapping but distinct
sets of their target genes (Figures 3 and 4). Regulators like
SKN-1 and DAF-16 affect longevity through control of numerous
genes that function in stress defense, metabolism, and other
processes (Kenyon, 2010). If these transcription factors influence overlapping biological processes, it could explain why
the life span extensions associated with genetic TORC1 and
TORC2 inhibition are not additive (Table S6), and why rapamycin
increases DAF-16 activity but extends life span independently of
DAF-16 (Figures 5, S4, and 6A). It will be an important challenge
to understand why the requirements for SKN-1 and DAF-16 differ
with respect to longevity associated with reductions in IIS,
TORC1, or TORC2 (Figure 7). This will involve identification of
critical downstream genes, and analyses of how these signaling
pathways (1) act in different tissues, (2) affect the action of
SKN-1, DAF-16, and possibly other transcription factors, and
(3) are influenced by other signals. For example, TORC2
and IIS activate kinases that regulate SKN-1 and DAF-16 (AKT
and SGK) (Kenyon, 2010; Zoncu et al., 2011) but seem to mirror
each other with respect to the relative importance of these transcription factors for longevity (Figure 7).
The TORC1 and IIS pathways involve an intricate set of autoand interregulatory networks. While we have shown that protective DAF-16/FoxO targets are activated when TORC1 is inhibited, in Drosophila and mammalian cells a feedback loop
has been described in which high TORC1 activity leads to accumulation of reactive oxygen species (ROS), and, in response,
FoxO increases expression of sestrins that inhibit TORC1 (Budanov et al., 2010; Hay, 2011). In mammals IIS increases TORC1
activity, but is inhibited by feedback loops emanating from
Cell Metabolism 15, 713–724, May 2, 2012 ª2012 Elsevier Inc. 721
Cell Metabolism
SKN-1, DAF-16, and TOR-Associated Longevity
Figure 7. Regulation of SKN-1 and DAF-16 by TOR Signaling
The IIS, TORC1, and TORC2 pathways are involved in growth (see the main
text). IIS inhibits SKN-1 and DAF-16 directly, through phosphorylation. TORC1
inhibits SKN-1 and DAF-16 expression and activity, at least in part by
increasing mRNA translation. TORC2 regulates SKN-1 nuclear occupancy in
a nutrient-dependent manner. DAF-16 is required for longevity that derives
from inhibition of IIS or TORC1, but not TORC2. SKN-1 plays a contributory
role in the effects of IIS on longevity but is essential for TORC1 or TORC2
inhibition to extend life span. When TORC1 is inhibited, SKN-1 increases
transcription of TORC1 pathway genes in a feedback loop.
TORC1 (Hsu et al., 2011; Yu et al., 2011; Zoncu et al., 2011).
When mammalian TORC1 activity is low, autophagy-driven
nutrient accumulation eventually drives a compensatory activation of TORC1 (Yu et al., 2010). We have described a feedback
loop that may help compensate for reduced TORC1 activity:
a SKN-1-driven induction of TORC1 pathway gene transcription
(Figure 7). These complex interregulatory loops may allow for
effective coordination of the TORC1 and IIS pathways with
each other, and with growth factor, ROS, and nutrient levels.
Several lines of evidence suggest that TORC1 regulates
SKN-1 and DAF-16 at least in part through modulating translation. Genetic inhibition of either TORC1 or translation initiation
resulted in similar effects on stress resistance and life span,
SKN-1 and DAF-16 nuclear occupancy, and regulation of downstream genes, including skn-1 and daf-16 themselves (see the
Results). Many genes encoding regulatory proteins are among
those that are translated preferentially when translation is globally reduced, and translation of both SKN-1 and DAF-16 seems
to be spared when translation initiation is suppressed (Kapahi
et al., 2010; Rogers et al., 2011; Stanfel et al., 2009; Zid et al.,
2009; Zoncu et al., 2011). We speculate that functional interactions among SKN-1, DAF-16, and other preferentially translated
proteins may lead to the transcriptional responses that result
from inhibiting either TORC1 or translation, although some of
the many phosphorylation targets of TORC1 could also be
involved (Hsu et al., 2011; Yu et al., 2011; Zoncu et al., 2011).
Our findings establish paradigms for understanding the biological effects of TORC1 signaling and rapamycin-related TOR
inhibitors. They predict that in mammals transcription programs
regulated by SKN-1/Nrf and DAF-16/FOXO may be important in
722 Cell Metabolism 15, 713–724, May 2, 2012 ª2012 Elsevier Inc.
the effects of TORC1, including its influence on stress resistance, growth, proliferation, development, stem cell function,
and TOR-associated diseases. Consistent with this idea, rapamycin treatment upregulated SKN-1/Nrf target genes in the
mouse, as seen in C. elegans (Figure 5). Our C. elegans evidence
indicated that rapamycin interferes with TORC2 in addition to
TORC1 in vivo (Figure 6), also in agreement with recent observations in the mouse (Lamming et al., 2012). In C. elegans, SKN-1
was required for longevity associated with genetic inhibition of
TORC1 or TORC2, or rapamycin treatment, strongly predicting
that Nrf proteins may mediate critical effects of rapamycin on
mammalian longevity. Development of agents that promote
particular SKN-1/Nrf or DAF-16/FoxO activities might therefore
capture beneficial effects of TOR inhibition without interfering
with critical TOR functions, or inducing unwanted effects of rapamycin on immune function or insulin sensitivity.
Together, our data show that the IIS and TOR pathways each
influence aging by regulating SKN-1 and DAF-16 (Figure 7).
Why would these pathways converge upon these two transcription factors? IIS, TORC1, and TORC2 are each involved in
growth, and SKN-1 and DAF-16 functions include maintenance
of protein homeostasis and resistance to various stresses (Kenyon, 2010; Lee et al., 2003; Li et al., 2011; McElwee et al., 2007;
Murphy et al., 2003; Oliveira et al., 2009). Under anabolic conditions it might be advantageous to inhibit these defenses, which
would influence regulatory processes in addition to consuming
resources. For example, IIS is suppressed by redox-regulated
phosphatases, and may function optimally under oxidizing conditions (Loh et al., 2009). Perhaps under conditions that do not favor
growth, such as poor nutrient availability, it is important to mobilize stress defenses that are regulated by SKN-1 and DAF-16. A
critical determinant of longevity seems to be the tradeoff between
growth and reproduction on one hand, and mechanisms that
increase stress resistance and delay aging on the other. Given
that the TOR and IIS pathways influence aging by regulating
SKN-1 and DAF-16, we conclude that the protective mechanisms
controlled by these two transcription factors are of central importance for the balance between growth signals and longevity.
EXPERIMENTAL PROCEDURES
Rapamycin Treatment of C. elegans
Rapamycin (LC laboratories) was dissolved in DMSO at 50 mg/ml and added
to plate agar to 100 mm unless otherwise indicated. Control plates contained
an appropriate DMSO concentration.
Life Span and Health Span
Life spans were carried out at 20 C essentially as described (Wang et al., 2010)
and were measured from hatching. RNAi or rapamycin treatments were
continued throughout life. Mitochondrial gene RNAi was initiated at the L2
stage. Health span assays were performed essentially as described (Huang
et al., 2004). Animals that crawled off the plate, ruptured, or died from internal
hatching were excluded.
ChIP
ChIP was performed as essentially as described (Glover-Cutter et al., 2008),
using a lysate protocol that was modified for C. elegans.
Transgenic Reporter Scoring
Expression or nuclear accumulation of transgenic GFP proteins was scored as
‘‘low,’’ ‘‘medium,’’ or ‘‘high’’ essentially as published (An and Blackwell, 2003;
Cell Metabolism
SKN-1, DAF-16, and TOR-Associated Longevity
Tullet et al., 2008; Wang et al., 2010). Details are in the Supplemental
Experimental Procedures.
Other methods are available in the Supplemental Experimental Procedures.
Hsu, P.P., Kang, S.A., Rameseder, J., Zhang, Y., Ottina, K.A., Lim, D.,
Peterson, T.R., Choi, Y., Gray, N.S., Yaffe, M.B., et al. (2011). The mTOR-regulated phosphoproteome reveals a mechanism of mTORC1-mediated inhibition
of growth factor signaling. Science 332, 1317–1322.
SUPPLEMENTAL INFORMATION
Huang, C., Xiong, C., and Kornfeld, K. (2004). Measurements of age-related
changes of physiological processes that predict lifespan of Caenorhabditis
elegans. Proc. Natl. Acad. Sci. USA 101, 8084–8089.
Supplemental Information includes Supplemental Experimental Procedures,
four figures, and seven tables and can be found with this article online at
doi:10.1016/j.cmet.2012.04.007.
ACKNOWLEDGMENTS
We thank Javier Apfeld, Eric Greer, Marcia Haigis, and the Blackwell lab for
helpful discussions, and David Bentley, Bruce Bowerman, and Heidi Tissenbaum for reagents. This work was supported by grants from the NIH
(GM62891 to T.K.B and CA129105 to D.M.S.), the Ellison Medical Foundation
(to T.K.B and to D.M.S.), the American Federation for Aging Research, Starr
Foundation, and Koch Institute Frontier Research Program (to D.M.S.), and
a DRC grant from the NIDDK. K.G.-C. was supported by an NIDDK T32 and
an F32 NRSA, D.W.L. by an F32 NRSA and a Mentor-Based Fellowship from
the ADA, and M.M. by the JSPS Excellent Young Researchers Overseas Visit
Program. T.K.B. planned the project, T.K.B. and D.M.S. oversaw the work, all
authors designed, carried out, and/or interpreted the experiments, and T.K.B.,
S.R.-S., K.G.-C., and D.W.L. wrote the paper.
Received: April 20, 2011
Revised: December 13, 2011
Accepted: April 6, 2012
Published online: May 1, 2012
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